Haspin is the prototypical member of a distinctive new group of serine/threonine kinases found throughout the eukaryotes. Haspin mRNA expression is highest in male germ cells and present at much lower levels in subsets of proliferating somatic cells. The protein is located in the nucleus of round and elongated spermatids and in a yet undefined nuclear sub-compartment, possibly the nucleolus, when transfected into cultured cells. Haspin protein levels appear to peak in haploid spermatids at a time when nucleoli undergo dissolution and inactivation. Over expression of haspin causes cell cycle arrest. It is therefore likely that haspin is involved in the cessation of meiosis in haploid germ cells. Regulation by and of haspin almost certainly involves both protein-protein interactions and phosphorylation events, activities commonly employed by critical regulators of cellular processes. Therefore, identification of haspin binding proteins will be crucial to understand its mechanism of action. Using a yeast-two hybrid screen of a human testis cDNA library, we have identified potential haspin binding proteins. Two of these candidates (Par4 and DEEPEST) bind to the N-terminus of haspin, while the other (Willbe) interacts with the C-terminal kinase domain. Par4 (prostate apoptosis response protein-4, also known as PRKC apoptosis WT1 regulator, PAWR) sensitizes cells to apoptosis, is a regulator of WT1 and has functional interactions with the protein kinases DIk/ZIP kinase and PKCzeta. DEEPEST (also known as Spag5, Astrin or hMAP126) is a recently described protein that is expressed predominantly in pachytene spermatocytes and round spermatids, but can also be incorporated into the mitotic spindle. Willbe is a previously uncharacterized protein with a similar tissue distribution to haspin. We will confirm these interactions in mammalian cells. To provide additional insight into the function of these proteins, we will determine the sub-nuclear localization of haspin and its binding partners. These studies of novel interactions between this intriguing kinase and other regulatory proteins should provide important insights into the control of the transition from meiosis to spermatid differentiation during spermatogenesis.